In April 1993 tuberculosis was declared as a global health emergency--the first such designation in the history of the World Health Organization. The distinction is regrettably justified because tuberculosis remains one of the largest burdens of disease and death in the world (Murray, C., et al. (1990) Bull. Int. Union Tuberc. Lung Disorders 65, 6-26; Murray, C., et al. (1997) in Global Burden of Diseases (Harvard Univ. Press, Cambridge, Mass.) p. 273) due in part to the increased susceptibility of HIV infected individuals and the ominous emergence of multi-drug resistant strains in both industrialized and developing countries. Effective new tuberculosis control and prevention strategies will require additional knowledge of the causative agent and its interaction with the human host. To systematically delineate virulence determinants, identify the metabolic pathways and discover novel drug targets for M. tuberculosis, a methodology generating libraries of mutants will be essential. Although mutant isolation and gene transfer strategies have been successfully employed for fast-growing non-virulent mycobacteria, such as M. smegmatis (Tokunaga, T., Mizuguchi, Y., & Suga, K. (1973) J. Bacteriol. 113, 1104-1111; Sudaraj, C. V. & Ramakrishnan, T. (1971) Nature (London) 228, 280-281), determining the genetic basis of phenotypes for M. tuberculosis has been frustrated by the lack of a natural gene transfer system in this pathogen. Furthermore, traditional mutational analyses based on the characterization of colonies arising from single cells following treatment with DNA damaging agents is of limited value for slow-growing mycobacteria, since the frequency of mutants is very low, multiple mutations occur in the same cells, and the mycobacteria tend to clump. Also, since M. tuberculosis or BCG have slow generation times (20 h-24 h), three to four weeks are required to visualize colonies arising from single cells, and many mutants are likely to grow even more slowly.
Transposon mutagenesis has been successfully used in diverse genera of bacteria (Berg, C. M., et al. (1989) In: Mobile DNA. American Society of Microbiology, Washington D.C. pp. 879-925). The first transposition events in M. smegmatis were reported using Tn610(Martin, C., et al. (1990) Nature (London) 21, 739-743), followed by transposons engineered from insertion elements IS900 and IS986 (Fomukong, N. G. & Dale, J. W. (1993) Gene 130, 99-105; England, P. M., Wall, Q., & McFadden, J. (1991) Mol. Microbiol. 5, 2047-2052). Transposition in BCG (McAdam, R.A., (1995) Infect. Immunol. 63 1004-1012) was reported using a transposon constructed from the insertion element IS1096 (Cirillo, J. D., et al. (1991) J. Bacteriol. 173, 7772-7780). Remarkably, the only reports of successful isolation of auxotrophic mutants for mycobacteria of the M. tuberculosis group have employed the use of insertional mutagenesis systems: illegitimate recombination (Kalpana, V. G., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 5433-5437), transposon mutagenesis (M.sup.c Adam, R. A., et al., (1995) Infect. Immun. 63, 1004-1012), and allelic exchange (Balasubramanian, V., et al. (1996) J. Bacteriol. 178, 273-279). A very promising approach to deliver transposons into M. smegmatis is the use of a conditionally replicating vector which is able to replicate at 30.degree. C., but not at 37.degree. C. (Guilhot, C., et al. (1994) J. Bacteriol. 176, 535-539). A library of 30,000 Tn611 insertion mutants was obtained from three independent experiments yielding 80 auxotrophic mutants with 15 different phenotypes. However, this system had not yet been applied to the slow-growing mycobacteria such as M. tuberculosis.
Conditionally replicating phage systems have proven to be very efficient systems for transposon mutagenesis in numerous bacterial species (Kleckner, N., et al. (1991) Methods Enzymol. 204 139-180). One of the great advantages of a phage delivery system is that essentially every cell in the bacterial population can be infected with the transposon-carrying phage, generating large numbers of independent mutants. Shuttle phasmid vectors, chimeric molecules that replicate in E. coli as plasmids and in mycobacteria as phages, were the first recombinant DNA vectors engineered for mycobacteria (Jacobs, Jr., W. R., Tuckman, M., & Bloom, B. R. (1987) Nature (London) 327, 532-536). Various phasmids, constructed from different mycobacteriophages, such as TM4, L1, and D29, have proven useful for the development of transformation systems for mycobacteria (Snapper, S. B., (1988) Proc. Natl. Acad. Sci. USA 85, 6987-6991) and the development of luciferase reporter phages for rapid diagnosis and drug susceptibility testing of M. tuberculosis clinical isolates (Jacobs, W. R., et al. (1993) Science 260, 819-822; Pearson, R. E., et al. (1996) Gene 183, 129-136).
Because the creation of mutants in M. tuberculosis and BCG is of essential importance in the analysis of gene function, it is desirable to develop effective means and methods for delivering foreign DNA into M. tuberculosis and BCG. The insertion of foreign DNA into M. tuberculosis and BCG mycobacteria would provide the necessary tools for understanding the mechanisms by which these mycobacteria survive and replicate. In addition, it would provide valuable tools for the development of vaccines and new drugs effective in the treatment of infection caused by M. tuberculosis and BCG.